Optogenetic Interrogation and Isolation of Photostimulated Neurons:
The function of a cortical neuron depends on its microcircuitry—the inputs it receives from local and long-range connections and the outputs it sends to other neurons. The existence of synaptic connections between two neurons can be inferred using neuroanatomical tracing but these methods cannot determine the frequency and strength of synaptic connections. Connectivity is often inferred during in vivo experiments by electrically or chemically stimulating a cluster of neurons and observing the effect on potential target neurons. However, both electrical and chemical stimulation indiscriminately activate a large group of neurons thus making it impossible to determine specific synaptic connections. In addition, cortical neurons receive inputs from the same source region via multiple pathways. Optogenetics can determine neural circuits with greater specificity and precision than previously possible. In particular, it has been recently demonstrated that viral vectors and in utero electroporation can be used to express light-sensitive channelrhodopsin-2 (ChR2) in a specific group of genetically determined neurons. Remarkably, when introduced in this manner, ChR2 is expressed not only near the cell bodies but is also transported to distant axon terminals. Injecting a viral vector carrying the gene for ChR2 into a cortical area resulted in the expression of ChR2 in axons within the thalamic projection zone of this cortical area. Photostimulation of these axons and the measurement of the photo-evoked activity then provide information about the direct influence exerted by this specific group of cortical neurons on their target neurons in this specific thalamic nucleus. Already, a handful of studies have used this technique to make exciting discoveries about specific brain areas that are involved in specific behaviors.
While great progress has been made in genetic methods used in optogenetics, little has been made in improving the devices used to simultaneously photostimulate and record neural activity (optrodes). These devices are made by either gluing microwires or electrodes to commercially available, thick optical fibers (e.g. ˜400-500 microns). To realize the full potential of optogenetic methods, a new type of optrode is required. This optrode should be capable of reaching many areas of interest buried within cortical sulci, deep subcortical structures like the amygdala, basal ganglia and the thalamus, as well as the brain stem. It should be therefore thin enough not to displace too much tissue or do much damage during repeated penetrations but at the same time it should be rigid enough to hit the target areas stereotaxically. It should be capable of delivering a significant amount of light power (˜100 mW) at its tip. Most important, it should provide electrodes with high signal-to-noise ratios and an optimized geometry for isolating single unit activity near the tip and in higher layers far from the region of direct photostimulation. The technology should include a simple method for interfacing the probes with standard neuroamplifier and signal processing systems and for fabricating linear and 2-D probe arrays. Finally, it should be compatible with automated manufacturing.
The design of a static probe with the 3-D mapping capability needed for the studies just described has been the subject of extensive theoretical and experimental investigation. Some have concluded that it will take 6 tiers of 4-channel sensors (tetrodes) on 10 μm pitch to acquire the rich data set needed to map the positions of neuronal dipoles with a precision equal to 10% of the recording volume. To access undisturbed tissue ahead of the probe and measure the activity of photostimulated neurons where the light field is most intense, each tetrode needs to be within about 60 micrometers of the tip. Thus, the setback of the first tetrode should be on the order of 10 μm. Moreover, the mapping of synchronous spike activity in ensemble studies may require electrode site diameters equal to or below about 5 μm. These specifications are far beyond the capability of current technology.
3-D Mapping of Neuronal Dipoles:
Modular organization is a hallmark of sensory cortex. Modules can be radial (e.g., orientation and ocular dominance columns, dendritic and pyramidal cell minicolumns) or tangential (e.g., cortical layers, cell rows). Driving inputs, feedback inputs, and outputs to a cortical neuron are largely determined by the layer in which the neuron is located. Neurons with similar functional properties are clustered into radial modules and arranged across the cortical surface in “functional maps.” Across species and cortical areas, estimated sizes of functional modules are 50 μm for a single orientation column in area V1 and in motor cortex; 250 μm for a cytochrome oxidase (CO) patch and color blobs in area V2; and 500 μm for an ocular dominance column in V1. Estimated sizes of anatomical modules are 22 μm for pyramidal cell modules in mouse barrel cortex; 60 μm in rat V1; 30-80 μm for ontogenic columns in various species; and 400-500 μm for projection fields or afferent columns in many species. Thus, local connections and functional properties of neurons can change in a few tens of microns. During extracellular recording of single or multiunit action potentials, the precise location of recorded neurons is unknown. As a result, sampled neurons have diverse functional properties and connectivity. When these neurons are pooled together and a hypothesis is tested, inconsistent results can be obtained depending on the chance constitution of the sample, generating controversies. Even basic questions about the size, constitution, and physiological properties of cortical minicolumns have not been settled 60 years after their discovery. A major impediment is the poor spatial resolution of the extracellular recording method. With such uncertain localization, it would be impossible to show that a receptive field property differs systematically from one minicolumn to the next. Such inherent limitations have plagued efforts to show that the minicolumn (or the column for that matter) has distinct properties and borders.
Extracellular recording locations are marked with small electrical lesions of tissue which can be identified post mortem during histological analysis. Recent techniques for such ex vivo localization of electrode tips include improved electrical lesioning and fluorescent dye-coated electrodes. Lesions and fluorescent traces are typically 50-400 μm wide, yielding an accuracy of 25-200 μm. About 95% of recorded neurons are distributed over a 132 μm radius of the extracellular electrode. Therefore, even if the electrode tip were localized using a state-of-the-art method, there will still be an uncertainty of at least ˜(50+132)=˜182 μm in the actual spatial location of the spike source. This region of uncertainty can encompass several functional and anatomical minicolumns. Localization uncertainty can be reduced further by coupling a tip-locating method with a monopole or dipole source localization procedure as describe above, thereby reducing post mortem localization uncertainty to ˜75 μm.
A few methods for in vivo localization of electrode tips have been developed, such as stereo x-ray imaging, coregistration of CT and MRI images, and of photograph-MRI-radiographs. However, localization errors of these methods are estimated to range ˜150-1000 μm, too large for source localization methods to be useful. Clearly advances in real time tip localization would have a terrific impact since then the exact location of active neurons anywhere in the brain could be determined with ˜50 μm accuracy or better.
The probe technology discussed herein may be used for source localization in conjunction with ex vivo techniques (see Validation section below). Finally, electrode tips are sometimes localized either in vivo or ex vivo with respect to an optically imaged functional map. However, prior art methods are in need of probes capable of localizing spike sources accurately.
Consider a specific example of how our probes can resolve major controversies. Across the surface of area V1, neurons are arranged as an orderly map of preferred orientation with occasional singularities. Optical imaging has showed that preferred orientation continuously changes around the singularity point, with iso-orientation domains converging to the point like the vanes of a pinwheel. The organization of pinwheel centers remains controversial. The magnitude of the optically imaged orientation signal at pinwheel centers is quite low. This could be due to either the pinwheel-center neurons being inherently poorly tuned for orientation or the orientation preference of pinwheel-center neurons changing abruptly in a very short distance, thereby smearing the optical signal. Single and multi-electrode array recordings, optical and 2-photon imaging, and other methods have yielded contradictory evidence. Some studies concluded that selectivity for orientation near pinwheels is sharp, others that it is broad, and yet others that there is no consistent relationship between pinwheel structures and orientation selectivity. This disagreement stems from the spatial localization error of current mapping methods. Typically, 6-8 orientation samples are used to estimate the preferred orientation of a neuron.
Based on the conservative estimate of 75 μm spatial resolution for our probes, we can deduce that neurons can be densely sampled and selectively assigned to 6-8 confidence bands, each 75 μm wide, around a pinwheel center. This should allow the precise mapping of orientation preference as close as ˜70 μm from the exact pinwheel center (i.e., [75*6/(2*π)]). As this example illustrates, our probes will help advance systems neuroscience by enabling the mapping of structure-function relationship in the cortex with significantly improved precision.
Multi-tiered neural probes and methods for manufacturing such probes are discussed herein. Systems and methods discussed herein integrate thin film electrodes and associated interconnect wiring on the cylindrical surface of optical fibers and other cylindrical or multi-sided wire substrates. In some embodiments, the neural probe may be an optrode and/or a tetrode. The neural probes may enable users to optically stimulate the axonal arbors of a specific group of genetically determined neurons, however deep in the brain these arbors might be, and record spikes from activated neurons in the vicinity of the fiber tip as well as at higher levels along the probe shank. Furthermore, dense arrays of such fine optrodes will enable neuroscientists to independently manipulate neurons at multiple sites with high spatial and temporal resolution. These advances may allow systems neuroscientists to establish causal relationships between different groups of neurons and map brain circuits with unprecedented specificity and precision.